The Rho GTPase-activating protein DLC1 is a tumor suppressor that is often deleted in liver cancer and downregulated in other cancers. DLC1 regulates the actin cytoskeleton, cell shape, adhesion, migration, and proliferation through its Rho GTPase-activating protein activity and focal adhesion localization. In this study, we silenced DLC1 in nonmalignant prostate epithelial cells to explore its tumor suppression functions. Small hairpin RNA-mediated silencing of DLC1 was insufficient to promote more aggressive phenotypes associated with tumor cell growth. In contrast, DLC1 silencing promoted pro-angiogenic responses through vascular endothelial growth factor (VEGF) upregulation, accompanied by the accumulation of hypoxia-inducible factor 1α and its nuclear localization. Notably, modulation of VEGF expression by DLC1 was dependent on epidermal growth factor receptor–MAP/ERK kinase–hypoxia-inducible factor 1 signaling but on RhoA pathways. Clinically, VEGF upregulation is a highly significant event in prostate cancers in which DLC1 is downregulated. Thus, our results strongly suggest that loss of DLC1 may serve as a “second hit” in promoting angiogenesis in a paracrine fashion during tumorigenesis. Cancer Res; 70(21); 8270–5. ©2010 AACR.

Deleted in liver cancer 1 (DLC1) is a tumor suppressor that was originally identified in primary hepatocellular carcinoma (1). In addition to liver cancer, the loss or reduction of DLC1 expression due to gene deletion or promoter methylation has been reported in lung, prostate, breast, kidney, colon, uterus, ovary, and stomach cancers (24). Mutations that altered the expression and function of DLC1 were detected in pancreas (5), colon, and prostate cancers (6). DLC1 is shown to regulate actin cytoskeleton and focal adhesion organizations, cell shape, adhesion, migration, proliferation, and apoptosis (24). These functions may directly contribute to the suppressive activities of DLC1 in tumorigenicity and metastasis (24). The RhoA pathway negatively regulated through the Rho GTPase-activating protein domain of DLC1 is believed to be critical for these functions, which were mainly analyzed by ectopic expression approaches in cancer cell lines.

Angiogenesis is the formation of new blood vessels from the existing vasculature and is essential for the growth of the primary cancer and for the formation of metastasis (7). Vascular endothelial growth factor (VEGF) plays a major role in tumor angiogenesis. It can promote the proliferation, survival, and migration of endothelial cells and is essential for blood vessel formation (8). Hypoxia is the strongest stimulus for triggering VEGF expression in cancer cells. Nonetheless, many cancer cell lines express high levels of VEGF in normoxia (9).

Here, we have discovered a novel function of DLC1 in regulating angiogenesis. Silencing of DLC1 in nonmalignant prostate epithelial cells leads to the upregulation of VEGF which promotes angiogenesis in vivo and in vitro. This upregulation of VEGF is mediated through the epidermal growth factor receptor (EGFR)–MAP/ERK kinase (MEK)–hypoxia-inducible factor 1 (HIF1) pathway. Clinically, upregulation of VEGF is highly associated with decreased DLC1 in prostate cancer.

Cell culture and reagents

MLC-SV40 kindly provided by Dr. Johng Rhim (Center for Prostate Disease Research, Bethesda, MD; ref. 10), and RWPE-1 cells purchased from the American Type Culture Collection (CRL-11609) were cultured in keratinocyte serum-free medium (Invitrogen). Human vascular endothelium cells (HUVEC) from American Type Culture Collection (CRL-1730) were cultured in endothelial cell growth medium (Genlantis). Cell lines were used within 3 months after receipt or resuscitation of frozen aliquots. The authentication of these cell lines was assured by the provider by cytogenetic analysis. No additional test was done specifically for this study. Lipofectamine-2000 (Invitrogen) was used for transfections. Stable shGFP or shDLC1 cells were generated by infection with small hairpin RNA lentiviruses against GFP or DLC1 (Sigma-Aldrich), followed by puromycin (2.5 μg/mL) selection. ELISA kit (R&D) was used to determine VEGF levels in conditioned medium. RhoA activity was measured with RhoA Activation Assay Kit (Cytoskeleton).

Xenograft assay

Growth factor–reduced Matrigel (BD Biosciences) containing 60 units/mL of heparin (Sigma-Aldrich) was mixed with 2 × 106 cells, and s.c. injected into nude mice. After 5 days, cell plugs were harvested and embedded in optimal cutting temperature compound for immunohistochemical staining using CD31 antibody and VEGF antibody.

In vitro Matrigel angiogenesis assay

Growth factor–reduced Matrigel was used to coat a 96-well plate (50 μL/well) and HUVECs (20,000 cells/well) were seeded with conditioned medium (200 μL). After 4 hours of incubation, capillary–like structures were scored by measuring the lengths of tubules per field in each well at ×100 magnification with ImageJ software (NIH).

Aortic ring assay

Thoracic aortas from C57BL/6 mice were dissected and transferred to ice-cold PBS. The fat tissue was removed and 1-mm-long aortic rings were sectioned and embedded in growth factor–reduced Matrigel. Rings were cocultured with 500 μL of conditioned medium with or without 1 μg of anti-VEGF blocking antibody (R&D, clone 26503) for 8 days, and the outgrowth of endothelial tubes was counted.

Migration assay

HUVECs (80,000 cells) were added to the upper chamber in each transwell. Conditioned media (400 μL) were added to the lower chamber. Cells were fixed and stained 5 hours later. Cells migrated to the bottom surface were visualized microscopically and photographed. For VEGF blockade, conditioned medium was incubated with 1 μg of anti-VEGF blocking antibody (R&D) at room temperature for 1 hour prior to experiments.

Adenoviruses

Human DLC1 cDNA was subcloned into pENTR1A vector and the DLC1/pENTR clone was used in a site-directed recombination reaction to place DLC1 cDNA into the pAD/CMV/V5-DEST vector (Invitrogen). The adenoviral expression clone was transfected into 293A cells. After 10 to 12 days, the crude viral lysate was harvested and used for infection.

Immunohistochemical staining, scoring, and microvascular density counting

Prostate normal/cancer tissue arrays (Imgenex) were dewaxed and rehydrated. After antigen retrieval, endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 20 minutes followed by normal serum blocking. Slides were incubated at 4°C overnight with anti-DLC1 (1:50, clone 3; BD Biosciences), anti-VEGF (1:50, SC152; Santa Cruz Biotechnology), or anti-CD31 (1:200, 01951D; PharMingen). Signals were detected with Vectastain ABC Elite Kit (Vector Laboratories) and diaminobenzidine substrate. Slides were counterstained with hematoxylin. Images were observed by Zeiss Axioplan2 microscope. For microvascular density counting, the vessels were detected by CD31 immunostaining and mean values of the vessel count were calculated using the average of the three most intense vascularization areas at 200× magnification. Tissue array immunoreactivities were scored by the intensity of the staining (0, no staining; 1, weak; 2, moderate; 3, strong) and the percentage of stained cells (0, no staining; 1, 1–10%; 2, 10–50%; 3, 50–80%; 4, >80%). By multiplication of both values, a final score ranging between 0 and 12 was obtained.

Silencing of DLC1 in nonmalignant prostate epithelial cells does not promote tumorigenicity but does enhance angiogenesis

To investigate the loss of function of DLC1 in normal prostate epithelial cells, we have generated DLC1 knockdown (shDLC1) by small hairpin RNA in the nonmalignant prostate epithelial cell line, MLC-SV40. DLC1 protein level was significantly reduced in shDLC1 than control shGFP cells, and as expected, RhoA activity was enhanced due to the reduction of the negative regulator DLC1 (Fig. 1A). To examine whether the loss of DLC1 enhanced their tumorigenicity, we injected shDLC1 or shGFP cells into nude mice. None of them developed tumors during 3 months of observation (data not shown). Interestingly, we detected increased small blood vessels (CD31 positive) around the injected shDLC1 cells at 5 days postinjection, and this was confirmed by significantly higher microvascular density in shDLC1 (Fig. 1B and C). We have generated an additional pair of shDLC1 and shGFP in another nonmalignant prostate cell line, RWPE-1. Similar results were observed using this pair of RWPE-1 cells throughout this project (Fig. 1A) and only data from MLC-SV40 cells are shown. These results suggest that silencing of DLC1 did not promote the proliferation of prostate epithelial cells, but somehow, it enhanced the angiogenic responses of endothelial cells.

Figure 1.

Silencing of DLC1 in prostate epithelial cells promotes angiogenesis in xenograft assays. A, DLC1 and RhoA expression levels in indicated cell lines were analyzed by immunoblotting. The amounts of active GTP-bound Rho GTPases were determined by GST-RBD pulldown assay followed by anti-RhoA immunoblotting. B, cells were injected s.c. into nude mice. Cell plugs were removed 5 d postinjection and processed for immunohistochemical staining using anti-CD31 and anti-VEGF antibodies. C, microvascular density and VEGF levels were scored and representative immunohistochemistry images were shown. Arrow, CD31-positive cells; arrowhead, VEGF-positive cells, respectively (bar, 50 μm).

Figure 1.

Silencing of DLC1 in prostate epithelial cells promotes angiogenesis in xenograft assays. A, DLC1 and RhoA expression levels in indicated cell lines were analyzed by immunoblotting. The amounts of active GTP-bound Rho GTPases were determined by GST-RBD pulldown assay followed by anti-RhoA immunoblotting. B, cells were injected s.c. into nude mice. Cell plugs were removed 5 d postinjection and processed for immunohistochemical staining using anti-CD31 and anti-VEGF antibodies. C, microvascular density and VEGF levels were scored and representative immunohistochemistry images were shown. Arrow, CD31-positive cells; arrowhead, VEGF-positive cells, respectively (bar, 50 μm).

Close modal

DLC1 negatively regulates VEGF expression

Because VEGF is the major proangiogenic factor, we examined VEGF levels in xenografts by immunohistochemical staining and found significantly elevated VEGF staining in injected shDLC1 cells, which were accompanied by CD31-positive endothelial cells (Fig. 1B and C). In cultured cells, VEGF mRNA and protein levels were upregulated in shDLC1 cells and the conditioned medium from shDLC1 also contained higher levels of VEGF (Fig. 2A), confirming that lack of DLC1 promotes VEGF expression and secretion. Furthermore, shDLC1 conditioned medium enhanced angiogenic-related responses of endothelial cells by forming more tube-like networks, faster cell migration, and more sprouting of capillaries (Fig. 2B). These effects were significantly reduced when anti-VEGF blocking antibody was applied (Fig. 2B). However, adding a similar amount of recombinant VEGF (500 pg/mL) to fresh medium did not have the same effects as detected with shDLC1-conditioned medium, suggesting that either recombinant VEGF was not as potent as endogenous VEGF or that there might be additional factor(s) in the conditioned medium that also contribute to the observed effects. Re-expression of small hairpin RNA-resistant DLC1 in shDLC1 markedly suppressed VEGF expression (Fig. 2C). A similar effect was detected when re-expression of DLC1 in prostate cancer cell lines, including LnCap, DU145, and even VEGF low-expressing PC3 cells (Fig. 2C), confirming that DLC1 negatively regulates VEGF expression.

Figure 2.

DLC1 regulates the expression of functional VEGF. A, VEGF mRNA, total protein, and secreted protein levels in the MLC-SV40 system were determined by RT-PCR, immunoblot, and ELISA assays. B, representative images and results of HUVEC tube formation (top), aortic ring (middle), and migration (bottom) assays using conditioned media from shGFP, shDLC1, or shDLC1 incubated with anti-VEGF blocking antibody. C, VEGF levels of shDLC1, PC3, LnCap, and Du145 cells infected with adenovirus expressing LacZ (AdLacZ as control) or DLC1 (AdDLC1) were measured by ELISA. All data are mean SD from triplicate experiments (*, P < 0.05).

Figure 2.

DLC1 regulates the expression of functional VEGF. A, VEGF mRNA, total protein, and secreted protein levels in the MLC-SV40 system were determined by RT-PCR, immunoblot, and ELISA assays. B, representative images and results of HUVEC tube formation (top), aortic ring (middle), and migration (bottom) assays using conditioned media from shGFP, shDLC1, or shDLC1 incubated with anti-VEGF blocking antibody. C, VEGF levels of shDLC1, PC3, LnCap, and Du145 cells infected with adenovirus expressing LacZ (AdLacZ as control) or DLC1 (AdDLC1) were measured by ELISA. All data are mean SD from triplicate experiments (*, P < 0.05).

Close modal

DLC1-regulated VEGF expression is mediated through the EGFR-MEK-HIF1 pathway but not the RhoA pathway

Because Rho GTPase-activating protein activity is critical for a variety of regulatory functions of DLC1, including cell shape, migration, and tumorigenicity, we test whether VEGF upregulation in shDLC1 cells is regulated through the RhoA pathway. By overexpression of RhoA wild-type, constitutive active and dominant negative mutants in MLC-SV40 cells, or silencing of RhoA expression in shDLC1 cells, VEGF levels were not affected (Supplementary Fig. S1), suggesting that DLC1-mediated VEGF expression is not regulated by the RhoA pathway.

To investigate the potential pathway(s) involved, several pharmacologic inhibitors were used. Although Jak (AG490) inhibitor had no effect on VEGF expression, inhibitors to EGFR (AG1478, AG1517) and MEK (PD98059, U0126) markedly reduced VEGF expression in shDLC1 cells in a dose-dependent manner (Fig. 3A). These inhibitor dosages have no effect on cell proliferation (Supplementary Fig. S2). Because HIF1 is a major transcription factor that mediates VEGF expression, we measured the protein and subcellular localization of HIF1α. Indeed, HIF1α protein level was increased and accumulated in the nuclei of shDLC1 cells (Fig. 3B and C). Concomitantly, increased EGFR (Tyr1068), MEK (Ser217/221), and ERK (Thr202/Tyr204) phosphorylation levels were detected (Fig. 3B). In addition, HIF1α protein levels and nuclear localization were markedly reduced by EGFR or MEK inhibitors (Fig. 3B–D). Furthermore, silencing of EGFR significantly decreased MEK and ERK phosphorylation, as well as VEGF expression in shDLC1 (Fig. 3D). These data suggest that DLC1-mediated VEGF expression is modulated through the EGFR-MEK-HIF1 pathway.

Figure 3.

VEGF upregulation in shDLC1 cells is mediated through the EGFR-MEK-HIF1 pathway. A, VEGF levels in conditioned media from shDLC1 cells treated with the indicated inhibitors at various concentrations for 24 h were measured by ELISA. B, cell lysates were analyzed by immunoblotting with indicated antibodies. C, cells grown on coverslips treated with the indicated reagents for 24 h were labeled for HIF1α and nuclei (propidium iodide) and visualized with Zeiss LSM 510 confocal microscope (bar, 10 μm). D, VEGF levels in conditioned media from cells treated with control or EGFR small interfering RNAs for 24 h were determined. The levels of EGFR, phosphorylated ERK, phosphorylated MEK in these cells were detected by immunoblotting (*, P < 0.05).

Figure 3.

VEGF upregulation in shDLC1 cells is mediated through the EGFR-MEK-HIF1 pathway. A, VEGF levels in conditioned media from shDLC1 cells treated with the indicated inhibitors at various concentrations for 24 h were measured by ELISA. B, cell lysates were analyzed by immunoblotting with indicated antibodies. C, cells grown on coverslips treated with the indicated reagents for 24 h were labeled for HIF1α and nuclei (propidium iodide) and visualized with Zeiss LSM 510 confocal microscope (bar, 10 μm). D, VEGF levels in conditioned media from cells treated with control or EGFR small interfering RNAs for 24 h were determined. The levels of EGFR, phosphorylated ERK, phosphorylated MEK in these cells were detected by immunoblotting (*, P < 0.05).

Close modal

Downregulation of DLC1 is associated with upregulation of VEGF in prostate cancer

To examine DLC1 and VEGF expression patterns in clinical samples, we have stained DLC1 and VEGF in consecutive prostate tissue sections. By immunohistochemical analysis, DLC1 is highly expressed in normal prostate epithelial cells, whereas VEGF expression is very weak, if present (Supplementary Fig. S3). In prostate cancer specimens, DLC1 expression was reduced in 86.3% (107 of 124), whereas VEGF protein level was increased in 75.8% (94 of 124) of tumor samples (Table 1). When comparing the expression of VEGF in DLC1 downregulated versus no change prostate cancer samples, increased VEGF expression is a statistically significant event in DLC1 downregulated prostate cancer.

Table 1.

DLC1 and VEGF expressions in prostate cancers

Total, 124 casesVEGF up (94)VEGF down or no change (30)
DLC1 down (107) 78/124 (62.9%) 29/124 (23.4%) 
DLC1 up or no change (17) 16/124 (12.9%) 1/124 (0.8%) 
Total, 124 casesVEGF up (94)VEGF down or no change (30)
DLC1 down (107) 78/124 (62.9%) 29/124 (23.4%) 
DLC1 up or no change (17) 16/124 (12.9%) 1/124 (0.8%) 

NOTE: Immunohistochemical analysis comparing prostate cancer to normal tissues indicated that increased VEGF expression in DLC1 downregulated prostate cancer was statistically significant. Fisher's exact test (P = 0.046).

Our current studies show that loss of DLC1 tumor suppressor alone is not sufficient for tumorigenesis. This is in agreement with the finding that DLC1 knockdown had little effect on the colony formation of liver progenitor cells in vitro but had efficiently promoted tumor development in Myc expressing and lacking p53 liver progenitor cells engrafted into mouse livers (11). Although lack of DLC1 does not increase cell tumorigenicity, it enhances angiogenesis, which plays an essential role in cancer progression. Tumor growth and metastasis are dependent on angiogenesis, and the preventions of tumor angiogenesis and/or metastasis are perceived as attractive approaches in the regulation of tumor progression. Because DLC1 also prevents cancer cell migration and metastasis (12, 13), understanding various DLC1-mediated pathways might offer potential targets for both angiogenesis and metastasis by enhancing the function of DLC1 or by suppressing its negatively regulated downstream molecules. Interestingly, our recent finding (14) showed that DLC2 knockout mice display enhanced angiogenic responses induced by Matrigel or tumor cells. These data suggest that the DLC family may play various roles in angiogenesis.

Other focal adhesion molecules may regulate VEGF expression. For example, overexpression of integrin-linked kinase stimulates VEGF expression, whereas silencing of integrin-linked kinase reduces VEGF production in prostate cancer cells (15). TM4SF5, a transmembrane protein that interacts with integrins, regulates VEGF expression through an integrin α5-Src-STAT3 pathway (16). Both integrin-linked kinase and TM4SF5 positively regulate VEGF expression, whereas DLC1 negatively controls VEGF production. This negative regulation of DLC1 seems to be mediated throughout EGFR signaling, which is known to regulate the synthesis and secretion of VEGF in cancer cells. Maity and colleagues (17) showed that EGFR activation regulates VEGF in glioblastoma cells through the phosphoinositide-3-kinase pathway but not the mitogen-activated protein kinase pathway. In head and neck squamous carcinoma, it is dependent on MEK/mitogen-activated protein kinase but not phosphoinositide-3-kinase pathways (18). These findings suggest that different mechanisms are involved in the ability of EGFR signaling in modulating VEGF expression in various types of cancer cells. Our data indicate that lack of DLC1 upregulates VEGF in an EGFR-MEK-HIF1–dependent fashion. Currently, we are investigating how and whether DLC1 directly or indirectly regulates the activity of EGFR.

No potential conflicts of interest were disclosed.

Grant Support: NIH grants CA102537 and CA151366 (S.H. Lo).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Yuan
BZ
,
Miller
MJ
,
Keck
CL
,
Zimonjic
DB
,
Thorgeirsson
SS
,
Popescu
NC
. 
Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP
.
Cancer Res
1998
;
58
:
2196
9
.
2
Liao
YC
,
Lo
SH
. 
Deleted in liver cancer-1 (DLC-1): a tumor suppressor not just for liver
.
Int J Biochem Cell Biol
2008
;
40
:
843
7
.
3
Durkin
ME
,
Yuan
BZ
,
Zhou
X
, et al
. 
DLC-1: a Rho GTPase-activating protein and tumour suppressor
.
J Cell Mol Med
2007
;
11
:
1185
207
.
4
Kim
TY
,
Vigil
D
,
Der
CJ
,
Juliano
RL
. 
Role of DLC-1, a tumor suppressor protein with RhoGAP activity, in regulation of the cytoskeleton and cell motility
.
Cancer Metastasis Rev
2009
;
28
:
77
83
.
5
Jones
S
,
Zhang
X
,
Parsons
DW
, et al
. 
Core signaling pathways in human pancreatic cancers revealed by global genomic analyses
.
Science
2008
;
321
:
1801
6
.
6
Liao
YC
,
Shih
YP
,
Lo
SH
. 
Mutations in the focal adhesion targeting region of deleted in liver cancer-1 attenuate their expression and function
.
Cancer Res
2008
;
68
:
7718
22
.
7
Folkman
J
. 
Tumor angiogenesis: therapeutic implications
.
N Engl J Med
1971
;
285
:
1182
6
.
8
Ferrara
N
. 
VEGF and the quest for tumour angiogenesis factors
.
Nat Rev Cancer
2002
;
2
:
795
803
.
9
Li
YM
,
Zhou
BP
,
Deng
J
,
Pan
Y
,
Hay
N
,
Hung
MC
. 
A hypoxia-independent hypoxia-inducible factor-1 activation pathway induced by phosphatidylinositol-3 kinase/Akt in HER2 overexpressing cells
.
Cancer Res
2005
;
65
:
3257
63
.
10
Lee
M-S
,
Garkovenko
E
,
Yun
JS
, et al
. 
Characterization of adult human prostatic cells immortalized by polybrene-induced DNA transfection with a plasmid containing an origin-defective SV40 genome
.
Int J Oncol
1994
;
4
:
821
30
.
11
Xue
W
,
Krasnitz
A
,
Lucito
R
, et al
. 
DLC1 is a chromosome 8p tumor suppressor whose loss promotes hepatocellular carcinoma
.
Genes Dev
2008
;
22
:
1439
44
.
12
Dydensborg
AB
,
Rose
AA
,
Wilson
BJ
, et al
. 
GATA3 inhibits breast cancer growth and pulmonary breast cancer metastasis
.
Oncogene
2009
;
28
:
2634
42
.
13
Goodison
S
,
Yuan
J
,
Sloan
D
, et al
. 
The RhoGAP protein DLC-1 functions as a metastasis suppressor in breast cancer cells
.
Cancer Res
2005
;
65
:
6042
53
.
14
Lin
Y
,
Chen
NT
,
Shih
YP
,
Liao
YC
,
Xue
L
,
Lo
SH
. 
DLC2 modulates angiogenic responses in vascular endothelial cells by regulating cell attachment and migration
.
Oncogene
2010
;
29
:
3010
6
.
15
Tan
C
,
Cruet-Hennequart
S
,
Troussard
A
, et al
. 
Regulation of tumor angiogenesis by integrin-linked kinase (ILK)
.
Cancer Cell
2004
;
5
:
79
90
.
16
Choi
S
,
Lee
SA
,
Kwak
TK
, et al
. 
Cooperation between integrin α5 and tetraspan TM4SF5 regulates VEGF-mediated angiogenic activity
.
Blood
2009
;
113
:
1845
55
.
17
Maity
A
,
Pore
N
,
Lee
J
,
Solomon
D
,
O'Rourke
DM
. 
Epidermal growth factor receptor transcriptionally up-regulates vascular endothelial growth factor expression in human glioblastoma cells via a pathway involving phosphatidylinositol 3′-kinase and distinct from that induced by hypoxia
.
Cancer Res
2000
;
60
:
5879
86
.
18
Bancroft
CC
,
Chen
Z
,
Yeh
J
, et al
. 
Effects of pharmacologic antagonists of epidermal growth factor receptor, PI3K and MEK signal kinases on NF-κB and AP-1 activation and IL-8 and VEGF expression in human head and neck squamous cell carcinoma lines
.
Int J Cancer
2002
;
99
:
538
48
.